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1.
Mol Cell ; 83(22): 4078-4092.e6, 2023 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-37977119

RESUMO

Tumor growth is driven by continued cellular growth and proliferation. Cyclin-dependent kinase 7's (CDK7) role in activating mitotic CDKs and global gene expression makes it therefore an attractive target for cancer therapies. However, what makes cancer cells particularly sensitive to CDK7 inhibition (CDK7i) remains unclear. Here, we address this question. We show that CDK7i, by samuraciclib, induces a permanent cell-cycle exit, known as senescence, without promoting DNA damage signaling or cell death. A chemogenetic genome-wide CRISPR knockout screen identified that active mTOR (mammalian target of rapamycin) signaling promotes samuraciclib-induced senescence. mTOR inhibition decreases samuraciclib sensitivity, and increased mTOR-dependent growth signaling correlates with sensitivity in cancer cell lines. Reverting a growth-promoting mutation in PIK3CA to wild type decreases sensitivity to CDK7i. Our work establishes that enhanced growth alone promotes CDK7i sensitivity, providing an explanation for why some cancers are more sensitive to CDK inhibition than normally growing cells.


Assuntos
Quinases Ciclina-Dependentes , Neoplasias , Humanos , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Quinase Ativadora de Quinase Dependente de Ciclina , Transdução de Sinais , Ciclo Celular , Inibidores Enzimáticos , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Linhagem Celular Tumoral
2.
PLoS Genet ; 19(8): e1010903, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37639469

RESUMO

Polo-like kinase 1 (PLK1) is a serine/threonine kinase required for mitosis and cytokinesis. As cancer cells are often hypersensitive to partial PLK1 inactivation, chemical inhibitors of PLK1 have been developed and tested in clinical trials. However, these small molecule inhibitors alone are not completely effective. PLK1 promotes numerous molecular and cellular events in the cell division cycle and it is unclear which of these events most crucially depend on PLK1 activity. We used a CRISPR-based genome-wide screening strategy to identify genes whose inactivation enhances cell proliferation defects upon partial chemical inhibition of PLK1. Genes identified encode proteins that are functionally linked to PLK1 in multiple ways, most notably factors that promote centromere and kinetochore function. Loss of the kinesin KIF18A or the outer kinetochore protein SKA1 in PLK1-compromised cells resulted in mitotic defects, activation of the spindle assembly checkpoint and nuclear reassembly defects. We also show that PLK1-dependent CENP-A loading at centromeres is extremely sensitive to partial PLK1 inhibition. Our results suggest that partial inhibition of PLK1 compromises the integrity and function of the centromere/kinetochore complex, rendering cells hypersensitive to different kinetochore perturbations. We propose that KIF18A is a promising target for combinatorial therapies with PLK1 inhibitors.


Assuntos
Proteínas de Ciclo Celular , Elementos Facilitadores Genéticos , Cinetocoros , Proteínas Serina-Treonina Quinases , Proteínas de Ciclo Celular/genética , Proteínas Serina-Treonina Quinases/genética , Humanos , Quinase 1 Polo-Like
3.
J Cell Biol ; 221(11)2022 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-36305789

RESUMO

Viruses co-opt host proteins to carry out their lifecycle. Repurposed host proteins may thus become functionally compromised; a situation analogous to a loss-of-function mutation. We term such host proteins as viral-induced hypomorphs. Cells bearing cancer driver loss-of-function mutations have successfully been targeted with drugs perturbing proteins encoded by the synthetic lethal (SL) partners of cancer-specific mutations. Similarly, SL interactions of viral-induced hypomorphs can potentially be targeted as host-based antiviral therapeutics. Here, we use GBF1, which supports the infection of many RNA viruses, as a proof-of-concept. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. Screening for SL partners of GBF1 revealed ARF1 as the top hit, disruption of which selectively killed cells that synthesize 3A alone or in the context of a poliovirus replicon. Thus, viral protein interactions can induce hypomorphs that render host cells selectively vulnerable to perturbations that leave uninfected cells otherwise unscathed. Exploiting viral-induced vulnerabilities could lead to broad-spectrum antivirals for many viruses, including SARS-CoV-2.


Assuntos
Fatores de Troca do Nucleotídeo Guanina , Poliovirus , Proteínas do Core Viral , Humanos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mutações Sintéticas Letais , Replicação Viral , Regulação Viral da Expressão Gênica , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Interações Hospedeiro-Patógeno
4.
Blood Adv ; 6(2): 509-514, 2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-34731885

RESUMO

Cholesterol homeostasis has been proposed as one mechanism contributing to chemoresistance in AML and hence, inclusion of statins in therapeutic regimens as part of clinical trials in AML has shown encouraging results. Chemical screening of primary human AML specimens by our group led to the identification of lipophilic statins as potent inhibitors of AMLs from a wide range of cytogenetic groups. Genetic screening to identify modulators of the statin response uncovered the role of protein geranylgeranylation and of RAB proteins, coordinating various aspect of vesicular trafficking, in mediating the effects of statins on AML cell viability. We further show that statins can inhibit vesicle-mediated transport in primary human specimens, and that statins sensitive samples show expression signatures reminiscent of enhanced vesicular trafficking. Overall, this study sheds light into the mechanism of action of statins in AML and identifies a novel vulnerability for cytogenetically diverse AML.


Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Leucemia Mieloide Aguda , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética
5.
Sci Adv ; 7(44): eabi5797, 2021 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-34705497

RESUMO

Pharmacological control of the ubiquitin-proteasome system (UPS) is of intense interest in drug discovery. Here, we report the development of chemical inhibitors of the ubiquitin-conjugating (E2) enzyme CDC34A (also known as UBE2R1), which donates activated ubiquitin to the cullin-RING ligase (CRL) family of ubiquitin ligase (E3) enzymes. A FRET-based interaction assay was used to screen for novel compounds that stabilize the noncovalent complex between CDC34A and ubiquitin, and thereby inhibit the CDC34A catalytic cycle. An isonipecotamide hit compound was elaborated into analogs with ~1000-fold increased potency in stabilizing the CDC34A-ubiquitin complex. These analogs specifically inhibited CDC34A-dependent ubiquitination in vitro and stabilized an E2~ubiquitin thioester reaction intermediate in cells. The x-ray crystal structure of a CDC34A-ubiquitin-inhibitor complex uncovered the basis for analog structure-activity relationships. The development of chemical stabilizers of the CDC34A-ubiquitin complex illustrates a general strategy for de novo discovery of molecular glue compounds that stabilize weak protein interactions.

6.
Aging Cell ; 20(4): e13331, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33660365

RESUMO

Telomere erosion in cells with insufficient levels of the telomerase reverse transcriptase (TERT), contributes to age-associated tissue dysfunction and senescence, and p53 plays a crucial role in this response. We undertook a genome-wide CRISPR screen to identify gene deletions that sensitized p53-positive human cells to telomerase inhibition. We uncovered a previously unannotated gene, C16ORF72, which we term Telomere Attrition and p53 Response 1 (TAPR1), that exhibited a synthetic-sick relationship with TERT loss. A subsequent genome-wide CRISPR screen in TAPR1-disrupted cells reciprocally identified TERT as a sensitizing gene deletion. Cells lacking TAPR1 or TERT possessed elevated p53 levels and transcriptional signatures consistent with p53 upregulation. The elevated p53 response in TERT- or TAPR1-deficient cells was exacerbated by treatment with the MDM2 inhibitor and p53 stabilizer nutlin-3a and coincided with a further reduction in cell fitness. Importantly, the sensitivity to treatment with nutlin-3a in TERT- or TAPR1-deficient cells was rescued by loss of p53. These data suggest that TAPR1 buffers against the deleterious consequences of telomere erosion or DNA damage by constraining p53. These findings identify C16ORF72/TAPR1 as new regulator at the nexus of telomere integrity and p53 regulation.


Assuntos
Aminobenzoatos , Peptídeos e Proteínas de Sinalização Intercelular , Naftalenos , Leucemia-Linfoma Linfoblástico de Células Precursoras , Transdução de Sinais , Telomerase , Proteína Supressora de Tumor p53 , Humanos , Aminobenzoatos/farmacologia , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Técnicas de Inativação de Genes , Imidazóis/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Naftalenos/farmacologia , Piperazinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Telomerase/antagonistas & inibidores , Telomerase/genética , Telomerase/metabolismo , Telômero/metabolismo , Transdução Genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/genética
7.
Cell Stem Cell ; 28(1): 48-62.e6, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33417871

RESUMO

Human hematopoietic stem cells (HSCs) exhibit attrition of their self-renewal capacity when cultured ex vivo, a process that is partially reversed upon treatment with epigenetic modifiers, most notably inhibitors of histone deacetylases (HDACs) or lysine-specific demethylase LSD1. A recent study showed that the human HSC self-renewal agonist UM171 modulates the CoREST complex, leading to LSD1 degradation, whose inhibition mimics the activity of UM171. The mechanism underlying the UM171-mediated loss of CoREST function remains undetermined. We now report that UM171 potentiates the activity of a CULLIN3-E3 ubiquitin ligase (CRL3) complex whose target specificity is dictated by the poorly characterized Kelch/BTB domain protein KBTBD4. CRL3KBTBD4 targets components of the LSD1/RCOR1 corepressor complex for proteasomal degradation, hence re-establishing H3K4me2 and H3K27ac epigenetic marks, which are rapidly decreased upon ex vivo culture of human HSCs.


Assuntos
Proteínas Correpressoras , Epigênese Genética , Células-Tronco Hematopoéticas , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Histona Desacetilases/metabolismo , Humanos
8.
bioRxiv ; 221(11)2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33173868

RESUMO

Viruses co-opt host proteins to carry out their lifecycle. Repurposed host proteins may thus become functionally compromised; a situation analogous to a loss-of-function mutation. We term such host proteins viral-induced hypomorphs. Cells bearing cancer driver loss-of-function mutations have successfully been targeted with drugs perturbing proteins encoded by the synthetic lethal partners of cancer-specific mutations. Synthetic lethal interactions of viral-induced hypomorphs have the potential to be similarly targeted for the development of host-based antiviral therapeutics. Here, we use GBF1, which supports the infection of many RNA viruses, as a proof-of-concept. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. Screening for synthetic lethal partners of GBF1 revealed ARF1 as the top hit, disruption of which, selectively killed cells that synthesize poliovirus 3A. Thus, viral protein interactions can induce hypomorphs that render host cells vulnerable to perturbations that leave uninfected cells intact. Exploiting viral-induced vulnerabilities could lead to broad-spectrum antivirals for many viruses, including SARS-CoV-2. SUMMARY: Using a viral-induced hypomorph of GBF1, Navare et al., demonstrate that the principle of synthetic lethality is a mechanism to selectively kill virus-infected cells.

9.
Mol Cell ; 79(5): 846-856.e8, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32755594

RESUMO

Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress.


Assuntos
Proliferação de Células/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Resveratrol/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular , Resistência a Medicamentos/genética , Humanos , Hidroxiureia/farmacologia , Células Jurkat , Nucleotídeos/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Sirtuína 1/metabolismo , Estilbenos/farmacologia
10.
Genetics ; 214(4): 1103-1120, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32094149

RESUMO

Systematic genetic interaction profiles can reveal the mechanisms-of-action of bioactive compounds. The imipridone ONC201, which is currently in cancer clinical trials, has been ascribed a variety of different targets. To investigate the genetic dependencies of imipridone action, we screened a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) knockout library in the presence of either ONC201 or its more potent analog ONC212. Loss of the mitochondrial matrix protease CLPP or the mitochondrial intermediate peptidase MIPEP conferred strong resistance to both compounds. Biochemical and surrogate genetic assays showed that impridones directly activate CLPP and that MIPEP is necessary for proteolytic maturation of CLPP into a catalytically competent form. Quantitative proteomic analysis of cells treated with ONC212 revealed degradation of many mitochondrial as well as nonmitochondrial proteins. Prompted by the conservation of ClpP from bacteria to humans, we found that the imipridones also activate ClpP from Escherichia coli, Bacillus subtilis, and Staphylococcus aureus in biochemical and genetic assays. ONC212 and acyldepsipeptide-4 (ADEP4), a known activator of bacterial ClpP, caused similar proteome-wide degradation profiles in S. aureus ONC212 suppressed the proliferation of a number of Gram-positive (S. aureus, B. subtilis, and Enterococcus faecium) and Gram-negative species (E. coli and Neisseria gonorrhoeae). Moreover, ONC212 enhanced the ability of rifampin to eradicate antibiotic-tolerant S. aureus persister cells. These results reveal the genetic dependencies of imipridone action in human cells and identify the imipridone scaffold as a new entry point for antibiotic development.


Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/agonistas , Imidazóis/farmacologia , Proteólise , Piridinas/farmacologia , Pirimidinas/farmacologia , Bacillus subtilis/efeitos dos fármacos , Sítios de Ligação , Sequência Conservada , Depsipeptídeos/metabolismo , Endopeptidase Clp/química , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , Metaloendopeptidases/metabolismo , Ligação Proteica , Rifampina/farmacologia , Staphylococcus aureus/efeitos dos fármacos
11.
Elife ; 82019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31868589

RESUMO

The cullin-RING ligases (CRLs) form the major family of E3 ubiquitin ligases. The prototypic CRLs in yeast, called SCF enzymes, employ a single E2 enzyme, Cdc34, to build poly-ubiquitin chains required for degradation. In contrast, six different human E2 and E3 enzyme activities, including Cdc34 orthologs UBE2R1 and UBE2R2, appear to mediate SCF-catalyzed substrate polyubiquitylation in vitro. The combinatorial interplay of these enzymes raises questions about genetic buffering of SCFs in human cells and challenges the dogma that E3s alone determine substrate specificity. To enable the quantitative comparisons of SCF-dependent ubiquitylation reactions with physiological enzyme concentrations, mass spectrometry was employed to estimate E2 and E3 levels in cells. In combination with UBE2R1/2, the E2 UBE2D3 and the E3 ARIH1 both promoted SCF-mediated polyubiquitylation in a substrate-specific fashion. Unexpectedly, UBE2R2 alone had negligible ubiquitylation activity at physiological concentrations and the ablation of UBE2R1/2 had no effect on the stability of SCF substrates in cells. A genome-wide CRISPR screen revealed that an additional E2 enzyme, UBE2G1, buffers against the loss of UBE2R1/2. UBE2G1 had robust in vitro chain extension activity with SCF, and UBE2G1 knockdown in cells lacking UBE2R1/2 resulted in stabilization of the SCF substrates p27 and CYCLIN E as well as the CUL2-RING ligase substrate HIF1α. The results demonstrate the human SCF enzyme system is diversified by association with multiple catalytic enzyme partners.


Assuntos
Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Genoma Humano/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Espectrometria de Massas , Poliubiquitina/genética , Transdução de Sinais/genética , Ubiquitinação/genética
12.
Cancer Cell ; 36(1): 84-99.e8, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31287994

RESUMO

To identify therapeutic targets in acute myeloid leukemia (AML), we chemically interrogated 200 sequenced primary specimens. Mubritinib, a known ERBB2 inhibitor, elicited strong anti-leukemic effects in vitro and in vivo. In the context of AML, mubritinib functions through ubiquinone-dependent inhibition of electron transport chain (ETC) complex I activity. Resistance to mubritinib characterized normal CD34+ hematopoietic cells and chemotherapy-sensitive AMLs, which displayed transcriptomic hallmarks of hypoxia. Conversely, sensitivity correlated with mitochondrial function-related gene expression levels and characterized a large subset of chemotherapy-resistant AMLs with oxidative phosphorylation (OXPHOS) hyperactivity. Altogether, our work thus identifies an ETC complex I inhibitor and reveals the genetic landscape of OXPHOS dependency in AML.


Assuntos
Antineoplásicos/farmacologia , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Oxazóis/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Triazóis/farmacologia , Animais , Biomarcadores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Hematopoese/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Camundongos , Modelos Biológicos , Receptor ErbB-2/antagonistas & inibidores
13.
Mol Cell Biol ; 38(1)2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-29038160

RESUMO

To interrogate genes essential for cell growth, proliferation and survival in human cells, we carried out a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 screen in a B-cell lymphoma line using a custom extended-knockout (EKO) library of 278,754 single-guide RNAs (sgRNAs) that targeted 19,084 RefSeq genes, 20,852 alternatively spliced exons, and 3,872 hypothetical genes. A new statistical analysis tool called robust analytics and normalization for knockout screens (RANKS) identified 2,280 essential genes, 234 of which were unique. Individual essential genes were validated experimentally and linked to ribosome biogenesis and stress responses. Essential genes exhibited a bimodal distribution across 10 different cell lines, consistent with a continuous variation in essentiality as a function of cell type. Genes essential in more lines had more severe fitness defects and encoded the evolutionarily conserved structural cores of protein complexes, whereas genes essential in fewer lines formed context-specific modules and encoded subunits at the periphery of essential complexes. The essentiality of individual protein residues across the proteome correlated with evolutionary conservation, structural burial, modular domains, and protein interaction interfaces. Many alternatively spliced exons in essential genes were dispensable and were enriched for disordered regions. Fitness defects were observed for 44 newly evolved hypothetical reading frames. These results illuminate the contextual nature and evolution of essential gene functions in human cells.


Assuntos
Sistemas CRISPR-Cas , Genes Essenciais/genética , Estudo de Associação Genômica Ampla/métodos , Proteoma/genética , Proteoma/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Biblioteca Gênica , Humanos , Proteômica/métodos
14.
Antioxid Redox Signal ; 28(1): 62-77, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-28398822

RESUMO

Precision in redox signaling is attained through posttranslational protein modifications such as oxidation of protein thiols. The peroxidase peroxiredoxin 1 (PRDX1) regulates signal transduction through changes in thiol oxidation of its cysteines. We demonstrate here that PRDX1 is a binding partner for the tumor suppressive transcription factor FOXO3 that directly regulates the FOXO3 stress response. Heightened oxidative stress evokes formation of disulfide-bound heterotrimers linking dimeric PRDX1 to monomeric FOXO3. Absence of PRDX1 enhances FOXO3 nuclear localization and transcription that are dependent on the presence of Cys31 or Cys150 within FOXO3. Notably, FOXO3-T32 phosphorylation is constitutively enhanced in these mutants, but nuclear translocation of mutant FOXO3 is restored with PI3K inhibition. Here we show that on H2O2 exposure, transcription of tumor suppressive miRNAs let-7b and let-7c is regulated by FOXO3 or PRDX1 expression levels and that let-7c is a novel target for FOXO3. Conjointly, inhibition of let-7 microRNAs increases let-7-phenotypes in PRDX1-deficient breast cancer cells. Altogether, these data ascertain the existence of an H2O2-sensitive PRDX1-FOXO3 signaling axis that fine tunes FOXO3 activity toward the transcription of gene targets in response to oxidative stress. Antioxid. Redox Signal. 28, 62-77.


Assuntos
Proteína Forkhead Box O3/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Oxirredução , Peroxirredoxinas/metabolismo , Interferência de RNA , Sítios de Ligação , Linhagem Celular , Movimento Celular , Dissulfetos , Humanos , Modelos Biológicos , Estresse Oxidativo , Peroxirredoxinas/genética , Regiões Promotoras Genéticas , Ligação Proteica , Transporte Proteico
15.
PLoS One ; 8(10): e77390, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24155950

RESUMO

Emergence of resistance to Tyrosine-Kinase Inhibitors (TKIs), such as imatinib, dasatinib and nilotinib, in Chronic Myelogenous Leukemia (CML) demands new therapeutic strategies. We and others have previously established bortezomib, a selective proteasome inhibitor, as an important potential treatment in CML. Here we show that the combined regimens of bortezomib with mitotic inhibitors, such as the microtubule-stabilizing agent Paclitaxel and the PLK1 inhibitor BI2536, efficiently kill TKIs-resistant and -sensitive Bcr-Abl-positive leukemic cells. Combined treatment activates caspases 8, 9 and 3, which correlate with caspase-induced PARP cleavage. These effects are associated with a marked increase in activation of the stress-related MAP kinases p38MAPK and JNK. Interestingly, combined treatment induces a marked decrease in the total and phosphorylated Bcr-Abl protein levels, and inhibits signaling pathways downstream of Bcr-Abl: downregulation of STAT3 and STAT5 phosphorylation and/or total levels and a decrease in phosphorylation of the Bcr-Abl-associated proteins CrkL and Lyn. Moreover, we found that other mitotic inhibitors (Vincristine and Docetaxel), in combination with bortezomib, also suppress the Bcr-Abl-induced pro-survival signals and result in caspase 3 activation. These results open novel possibilities for the treatment of Bcr-Abl-positive leukemias, especially in the imatinib, dasatinib and nilotinib-resistant CML cases.


Assuntos
Ácidos Borônicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Fusão bcr-abl/metabolismo , Leucemia/patologia , Mitose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/farmacologia , Benzamidas/farmacologia , Bortezomib , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dasatinibe , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Compostos Heterocíclicos com 2 Anéis/farmacologia , Humanos , Mesilato de Imatinib , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Leucemia/enzimologia , Modelos Biológicos , Paclitaxel/farmacologia , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Pteridinas , Pirimidinas/farmacologia , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
16.
Apoptosis ; 18(10): 1154-62, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23917691

RESUMO

Successful translation of findings derived from preclinical studies into effective therapies is critical in biomedical research. Lack of robustness and reproducibility of the preclinical data, due to insufficient number of repeats, inadequate cell-based and mouse models contribute to the poor success rate. Antibodies are widely used in preclinical research, notably to determine the expression of potential therapeutic targets in tissues of interest, including tumors, but also to identify disease and/or treatment response biomarkers. We sought to determine whether the current antibody characterization standards in preclinical research are sufficient to ensure reliability of the data found in peer-reviewed publications. To address this issue, we used detection of the protein c-FLIP, a major factor of resistance to apoptosis, as a proof of concept. Accurate detection of endogenous c-FLIP levels in the preclinical settings is imperative since it is considered as a potential theranostic biomarker. Several sources of c-FLIP antibodies validated by their manufacturer and recommended for western blotting were therefore rigorously tested. We found a wide divergence in immune recognition properties. While these antibodies have been used in many publications, our results show that several of them failed to detect endogenous c-FLIP protein by Western blotting. Our results suggest that antibody validation standards are inadequate, and that systematic use of genetic knockdowns and/or knockouts to establish proof of specificity is critical, even for antibodies previously used in the scientific literature. Because antibodies are fundamental tools in both preclinical and clinical research, ensuring their specificity is crucial.


Assuntos
Anticorpos/imunologia , Especificidade de Anticorpos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/imunologia , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Linhagem Celular Tumoral , Humanos , Camundongos , Coelhos
17.
Proc Natl Acad Sci U S A ; 109(39): 15793-8, 2012 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-23019363

RESUMO

Dinoflagellates are an important component of the marine biota, but a large genome with high-copy number (up to 5,000) tandem gene arrays has made genomic sequencing problematic. More importantly, little is known about the expression and conservation of these unusual gene arrays. We assembled de novo a gene catalog of 74,655 contigs for the dinoflagellate Lingulodinium polyedrum from RNA-Seq (Illumina) reads. The catalog contains 93% of a Lingulodinium EST dataset deposited in GenBank and 94% of the enzymes in 16 primary metabolic KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, indicating it is a good representation of the transcriptome. Analysis of the catalog shows a marked underrepresentation of DNA-binding proteins and DNA-binding domains compared with other algae. Despite this, we found no evidence to support the proposal of polycistronic transcription, including a marked underrepresentation of sequences corresponding to the intergenic spacers of two tandem array genes. We also have used RNA-Seq to assess the degree of sequence conservation in tandem array genes and found their transcripts to be highly conserved. Interestingly, some of the sequences in the catalog have only bacterial homologs and are potential candidates for horizontal gene transfer. These presumably were transferred as single-copy genes, and because they are now all GC-rich, any derived from AT-rich contexts must have experienced extensive mutation. Our study not only has provided the most complete dinoflagellate gene catalog known to date, it has also exploited RNA-Seq to address fundamental issues in basic transcription mechanisms and sequence conservation in these algae.


Assuntos
Dinoflagellida/genética , Genes de Protozoários/fisiologia , RNA de Protozoário/genética , Sequência de Bases , Dinoflagellida/metabolismo , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA de Protozoário/metabolismo , Transcrição Gênica/fisiologia
18.
J Biol Rhythms ; 23(5): 400-8, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18838606

RESUMO

In many phytoplankton species, cell division (mitosis) usually occurs at defined times of day. This timing is also observed under constant conditions, indicating that it is regulated by a circadian clock rather than by a simple response to the light-dark cycle. For those algae with cell cycles longer than a day, the clock opens a window of opportunity for mitosis at a particular time of day through which cells in an appropriate phase of the cell cycle can pass. Although the timing of mitosis is generally studied due to ease of measurement, for some phytoplankton the timing of S-phase is also circadian. This thus raises the possibility that mitosis is not directly gated by the clock but occurs instead at a defined interval (a constant G2 length) following a circadian controlled S-phase. To determine if the clock exercises independent control over the timing of both S- and M-phase, we measured the timing of both S- and M-phase in cultures of the dinoflagellate Lingulodinium grown under a variety of different photoperiods. We interpret the phase angles of both rhythms, in particular those resulting in a change in the length of G2, as an indication that the clock independently regulates the timing of S-phase and mitosis.


Assuntos
Divisão Celular , Ritmo Circadiano/fisiologia , Dinoflagellida/metabolismo , Eucariotos/fisiologia , Fase S , Animais , Separação Celular , Citometria de Fluxo , Luz , Mitose , Modelos Biológicos , Fotoperíodo , Fitoplâncton/metabolismo , Fatores de Tempo
19.
Protist ; 158(4): 473-85, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17573241

RESUMO

An AAA protein from the dinoflagellate Gonyaulax polyedra (GpAAA) with the unusual ability to rescue the phenotype of a yeast mutant lacking G1/S phase cyclins (cln1cln2cln3) has been isolated and the mechanism of rescue was characterized. We find that GpAAA is not a cyclin and has no similarity to any known cell cycle regulators. Instead, GpAAA forms a novel and strongly supported clade with bacterial spoIIIAA proteins and an Arabidopsis gene of unknown function. Since dinoflagellates cannot be transformed, we took advantage of the powerful genetic tools available for yeast. We find that rescue of the cln1cln2cln3 phenotype does not involve an effect on the CDK-inhibitor (CKI) Sic1p, as GpAAA does not alter the sensitivity to an inducible SIC1. Instead, Northern blot analyses show that GpAAA expression increases levels of CLB5, in agreement with the observation that GpAAA is unable to rescue the quadruple mutant cln1cln2cln3clb5. We propose that the increased transcription of CLB5 may be due to a protein remodeling function of GpAAA alleviating inhibition of the transcription factor SBF. Thus, although no known equivalents to the yeast SBF have been documented in dinoflagellates, we conclude that dinoflagellates could indeed utilize GpAAA as a cell cycle regulator.


Assuntos
Ciclinas/genética , Dinoflagellida/genética , Mutação , Proteínas de Protozoários/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Animais , Arabidopsis/genética , Proteínas de Bactérias/genética , Northern Blotting , Ciclina B/genética , DNA de Protozoário/química , DNA de Protozoário/genética , Expressão Gênica , Genes Essenciais , Genes Fúngicos , Teste de Complementação Genética , Dados de Sequência Molecular , Proteínas de Plantas/genética , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
20.
Biol Cell ; 99(9): 531-40, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17451378

RESUMO

BACKGROUND INFORMATION: Mitosis during the dinoflagellate cell cycle is unusual in that the nuclear envelope remains intact and segregation of the permanently condensed chromosomes uses a cytoplasmic mitotic spindle. To examine regulation of the dinoflagellate cell cycle in the context of these unusual nuclear features, it is necessary to isolate and characterize cell cycle regulators such as CDK (cyclin-dependent kinase). RESULTS: We report the characterization of a CDK from the dinoflagellate Lingulodinium polyedrum. This CDK reacts with an anti-PSTAIRE antibody and was identified by protein microsequencing after partial purification. The protein microsequence shows homology toward the Pho85/CDK5 clade of CDKs. Neither the amount nor the phosphorylation state changed over the course of the cell cycle, in agreement with results reported for CDK5 family members in other systems. CONCLUSIONS: We conclude we have probably isolated a dinoflagellate CDK5-like protein. The data reported here support the identification of this protein as a CDK5 homologue, and suggest that dinoflagellates may contain several CDK families.


Assuntos
Quinase 5 Dependente de Ciclina/química , Dinoflagellida/química , Animais , Western Blotting , Ciclo Celular , Quinase 5 Dependente de Ciclina/classificação , Quinase 5 Dependente de Ciclina/isolamento & purificação , Quinases Ciclina-Dependentes/química , Citometria de Fluxo , Filogenia , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência
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